The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Peter Riederer - One of the best experts on this subject based on the ideXlab platform.
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Orphenadrine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist: binding and patch clamp studies
Journal of Neural Transmission, 1995Co-Authors: Johannes Kornhuber, John Thome, S. Kamolz, C. G. Parsons, W Retz, Stefan Hartmann, Peter RiedererAbstract:Orphenadrine has been used as an antiparkinsonian, antispastic and analgesic drug for many years. Here we show that orphenadrine inhibits [3H]MK-801 binding to the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA)-receptor in homogenates of postmortem human frontal cortex with a Ki-value of 6.0 +/- 0.7 microM. The NMDA receptor antagonistic effects of orphenadrine were assessed using concentration- and patch-clamp techniques on cultured superior colliculus neurones. Orphenadrine blocked open NMDA receptor channels with fast kinetics and in a strongly voltage-dependent manner. The IC50-value against steady state currents at -70 mV was 16.2 +/- 1.6 microM (n = 6). Orphenadrine exhibited relatively fast, concentration-dependent open channel blocking kinetics (Kon 0.013 +/- 0.002 10(6) M-1S-1) whereas the offset rate was concentration-independent (Koff 0.230 +/- 0.004 S-1). Calculation of the ratio Koff/Kon revealed an apparent Kd-value of 17.2 microM which is nearly identical to the IC50 calculated at equilibrium.
Masaharu Nakayama - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Kinetic Binding Properties of Unlabeled Ligands via a Preincubation Endpoint Binding Approach
Journal of Biomolecular Screening, 2016Co-Authors: Yuji Shimizu, Kazumasa Ogawa, Masaharu NakayamaAbstract:The dissociation rates of unlabeled drugs have been well studied by kinetic binding analyses. Since kinetic assays are laborious, we developed a simple method to determine the kinetic binding parameters of unlabeled competitors by a preincubation endpoint assay. The probe binding after preincubation of a competitor can be described by a single equation as a function of time. Simulations using the equation revealed the degree of IC50 change induced by preincubation of a competitor depended on the dissociation rate Koff of the competitor but not on the association rate kon. To validate the model, an in vitro binding assay was performed using a smoothened receptor (SMO) and [3H]TAK-441, a SMO antagonist. The equilibrium dissociation constants (KI) and Koff of SMO antagonists determined by globally fitting the model to the concentration–response curves obtained with and without 24 h preincubation correlated well with those determined by other methods. This approach could be useful for early-stage optimization...
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biochemical characterization of smoothened receptor antagonists by binding kinetics against drug resistant mutant
European Journal of Pharmacology, 2015Co-Authors: Yuji Shimizu, Tsuyoshi Ishii, Kazumasa Ogawa, Satoshi Sasaki, Hideki Matsui, Masaharu NakayamaAbstract:Abstract Hedgehog (Hh) signaling critical for development, differentiation, and cell growth is involved in several cancers, including medulloblastoma and basal cell carcinoma. Although antagonism of the smoothened receptor (SMO), which mediates Hh signaling, is an attractive therapeutic target, a drug-resistant mutation in SMO (SMO-D473H) was identified in a clinical trial of the approved drug vismodegib. TAK-441 potently inhibits SMO-D473H, unlike vismodegib and another SMO antagonist, cyclopamine, whereas the differences in binding modes between these antagonists remain unknown. Here we report the biochemical characterization of TAK-441, vismodegib, and cyclopamine by binding kinetics. The association (kon) and dissociation (Koff) rates were determined by kinetic binding studies using [3H]TAK-441, and dissociation was confirmed by label-free affinity selection-mass spectrometry (AS-MS). In the [3H]TAK-441 competition assay, TAK-441 but not vismodegib and cyclopamine showed time-dependent inhibition. Quantitative kinetic binding analysis revealed that Koff of TAK-441 was >10-fold smaller than those of vismodegib and cyclopamine. To further assess the binding mode of antagonists, kinetic binding analysis was performed against SMO-D473H. The D473H mutation affected Koff of TAK-441 but not kon. In contrast, only kon was changed by the D473H mutation in the case of vismodegib and cyclopamine. These results suggest that the difference in antagonist efficacy against D473H is associated with the binding mode of antagonists. These findings provide a new insight into the drug action of SMO antagonists and help develop potential therapeutics for drug-resistant mutants.
Johannes Kornhuber - One of the best experts on this subject based on the ideXlab platform.
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Orphenadrine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist: binding and patch clamp studies
Journal of Neural Transmission, 1995Co-Authors: Johannes Kornhuber, John Thome, S. Kamolz, C. G. Parsons, W Retz, Stefan Hartmann, Peter RiedererAbstract:Orphenadrine has been used as an antiparkinsonian, antispastic and analgesic drug for many years. Here we show that orphenadrine inhibits [3H]MK-801 binding to the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA)-receptor in homogenates of postmortem human frontal cortex with a Ki-value of 6.0 +/- 0.7 microM. The NMDA receptor antagonistic effects of orphenadrine were assessed using concentration- and patch-clamp techniques on cultured superior colliculus neurones. Orphenadrine blocked open NMDA receptor channels with fast kinetics and in a strongly voltage-dependent manner. The IC50-value against steady state currents at -70 mV was 16.2 +/- 1.6 microM (n = 6). Orphenadrine exhibited relatively fast, concentration-dependent open channel blocking kinetics (Kon 0.013 +/- 0.002 10(6) M-1S-1) whereas the offset rate was concentration-independent (Koff 0.230 +/- 0.004 S-1). Calculation of the ratio Koff/Kon revealed an apparent Kd-value of 17.2 microM which is nearly identical to the IC50 calculated at equilibrium.
Yuji Shimizu - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Kinetic Binding Properties of Unlabeled Ligands via a Preincubation Endpoint Binding Approach
Journal of Biomolecular Screening, 2016Co-Authors: Yuji Shimizu, Kazumasa Ogawa, Masaharu NakayamaAbstract:The dissociation rates of unlabeled drugs have been well studied by kinetic binding analyses. Since kinetic assays are laborious, we developed a simple method to determine the kinetic binding parameters of unlabeled competitors by a preincubation endpoint assay. The probe binding after preincubation of a competitor can be described by a single equation as a function of time. Simulations using the equation revealed the degree of IC50 change induced by preincubation of a competitor depended on the dissociation rate Koff of the competitor but not on the association rate kon. To validate the model, an in vitro binding assay was performed using a smoothened receptor (SMO) and [3H]TAK-441, a SMO antagonist. The equilibrium dissociation constants (KI) and Koff of SMO antagonists determined by globally fitting the model to the concentration–response curves obtained with and without 24 h preincubation correlated well with those determined by other methods. This approach could be useful for early-stage optimization...
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biochemical characterization of smoothened receptor antagonists by binding kinetics against drug resistant mutant
European Journal of Pharmacology, 2015Co-Authors: Yuji Shimizu, Tsuyoshi Ishii, Kazumasa Ogawa, Satoshi Sasaki, Hideki Matsui, Masaharu NakayamaAbstract:Abstract Hedgehog (Hh) signaling critical for development, differentiation, and cell growth is involved in several cancers, including medulloblastoma and basal cell carcinoma. Although antagonism of the smoothened receptor (SMO), which mediates Hh signaling, is an attractive therapeutic target, a drug-resistant mutation in SMO (SMO-D473H) was identified in a clinical trial of the approved drug vismodegib. TAK-441 potently inhibits SMO-D473H, unlike vismodegib and another SMO antagonist, cyclopamine, whereas the differences in binding modes between these antagonists remain unknown. Here we report the biochemical characterization of TAK-441, vismodegib, and cyclopamine by binding kinetics. The association (kon) and dissociation (Koff) rates were determined by kinetic binding studies using [3H]TAK-441, and dissociation was confirmed by label-free affinity selection-mass spectrometry (AS-MS). In the [3H]TAK-441 competition assay, TAK-441 but not vismodegib and cyclopamine showed time-dependent inhibition. Quantitative kinetic binding analysis revealed that Koff of TAK-441 was >10-fold smaller than those of vismodegib and cyclopamine. To further assess the binding mode of antagonists, kinetic binding analysis was performed against SMO-D473H. The D473H mutation affected Koff of TAK-441 but not kon. In contrast, only kon was changed by the D473H mutation in the case of vismodegib and cyclopamine. These results suggest that the difference in antagonist efficacy against D473H is associated with the binding mode of antagonists. These findings provide a new insight into the drug action of SMO antagonists and help develop potential therapeutics for drug-resistant mutants.
John Thome - One of the best experts on this subject based on the ideXlab platform.
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Orphenadrine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist: binding and patch clamp studies
Journal of Neural Transmission, 1995Co-Authors: Johannes Kornhuber, John Thome, S. Kamolz, C. G. Parsons, W Retz, Stefan Hartmann, Peter RiedererAbstract:Orphenadrine has been used as an antiparkinsonian, antispastic and analgesic drug for many years. Here we show that orphenadrine inhibits [3H]MK-801 binding to the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA)-receptor in homogenates of postmortem human frontal cortex with a Ki-value of 6.0 +/- 0.7 microM. The NMDA receptor antagonistic effects of orphenadrine were assessed using concentration- and patch-clamp techniques on cultured superior colliculus neurones. Orphenadrine blocked open NMDA receptor channels with fast kinetics and in a strongly voltage-dependent manner. The IC50-value against steady state currents at -70 mV was 16.2 +/- 1.6 microM (n = 6). Orphenadrine exhibited relatively fast, concentration-dependent open channel blocking kinetics (Kon 0.013 +/- 0.002 10(6) M-1S-1) whereas the offset rate was concentration-independent (Koff 0.230 +/- 0.004 S-1). Calculation of the ratio Koff/Kon revealed an apparent Kd-value of 17.2 microM which is nearly identical to the IC50 calculated at equilibrium.
